基于核酸适配体的各种传感技术近年来在真菌毒素高灵敏度检测中应用研究越来越多,但适配体结构的动态柔性特点极易导致假阳性及低识别能力的问题,影响了其实际应用。为了解决由于适配体柔性结构导致的假阳性问题,该文以黄曲霉毒素M1(aflatoxin M1, AFM1)为目标物,将整条适配体链劈裂成两条短链,减少适配体结构柔性,稳定其结构,构建基于劈裂适配体的双信号电化学传感器。研究中先将一段完整AFM1适配体劈裂成两段(S1和S2),其中一段(S1)修饰巯基,利用Au-S将其固定在电极表面,劈裂适配体的另一段(S2)修饰亚甲基蓝(methylene blue, MB)作为传感信号,另外S1的互补链CS1修饰二茂铁(ferrocene, Fc)作为另一传感信号。体系中没有AFM1时,S1与CS1互补形成双链结构,Fc信号有响应;当体系中添加AFM1后,双链解开,CS1释放, S1与S2形成一定构象共同识别AFM1,Fc信号减弱,MB信号增加。研究结果表明,该传感器检测AFM1的线性范围是0.050~0.800 μg/L,最低检出限为0.015 μg/L,同时具有良好的选择性。该方法为解决适配体稳定性问题提供一个新的思路。
Recently, various biosensors based on nucleic acid aptamers have been applied to the detection of mycotoxins with high sensitivity, but the dynamic flexibility of aptamer structure can easily lead to false positive and low recognition ability, which limits the application of aptamer sensing technology. To solve this problem, a complete aptamer was split into two aptamer fragments to reduce the structural flexibility of aptamers and stabilize its structure, and a dual-signal electrochemical sensor based on split aptamers was constructed for the detection of AFM1. In this work, AFM1 aptamer was split into two segments (S1 and S2). S1 was modified with the thiol group, and then was immobilized on the electrode surface by Au-S. S2 was modified with methylene blue (MB) as the sensing signal, and the complementary chain CS1 of S1 was modified with ferrocene (Fc) as another sensing signal. Without AFM1, a double chain structure was formed by S1 and CS1, and the Fc signal enhanced. In the presence of AFM1, CS1 was released, and S1 and S2 formed a certain conformation to recognize AFM1. The intensity of Fc decreased, and the intensity of MB increased. The results showed that the linear range of this sensor was 0.050-0.800 μg/L with the minimum detection limit of 0.015 μg/L, and the sensor had a good selectivity. This method provides a new idea to solve the problem of aptamer stability.
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